Enzymes that catalyze the synthesis, utilization, and interconversion of phosphorylated-metabolites usually require specific activation by inorganic cations, and reactions involving metal nucleotide substrates or effectors are of central importance in a wide varity of cellular processes. Understanding the roles of metal ions in the respective catalytic activations, and in the overall mechanisms for these reactions requires detailed information on the binding interactions that occur between the metal ion, substrates, and protein in the reactive complexes. The goals of this project are to determine the coordination schemes in enzyme-metal ion-substrate and inhibitor complexes in order to understand the relationships between specific binding interactions and chemical activation of these molecules and the strategies that are exploited in the catalytic mechanisms. The proposed research will use electron paramagnetic resonance, nuclear magnetic resonance, and vibrational spectroscopy, together with isotopically labeled molecules (substrates and analogue), to determine the binding schemes between metal ion, substrate and protein at the active sites of several enzymes that catalyze phosphotransfer reactions. The experiments will also provide information on the stereospecificities of enzyme active sites and on the mode of action of specific inhibitory compounds. Knowledge of the structures of enzyme-substrate complexes is essential to the rational design of specific inhibitors that can be used as therapeutic agents, or as biochemical and physiological reagents to control, selectively, reactions or metabolic pathways. The information is also of fundamental importance in understanding the mechanisms for biological catalysis.